Monitoring resistance to methomyl and synergism in the non-target Musca domestica from cotton fields of Punjab and Sindh provinces, Pakistan

Insecticides are an integral part of most of the cropping systems worldwide; however, these usually exert negative impact on the environment and non-target insects as well. Non-target insects are prone to develop resistance to insecticides due to prolonged and repeated lethal and sublethal exposures. Musca domestica is a common non-target, pollinator and nectar feeder species in cotton ecosystem, besides its status as a public health pest in human habitations. In the present work, resistance to methomyl, one of the major insecticides used for cotton pest management, was assessed in 20 M. domestica strains from the major cotton producing areas of the Punjab and Sindh provinces of Pakistan. The results revealed that toxicity values of methomyl for Punjabi and Sindhi strains ranged from 28.07 to 136.16 µg fly−1 and 29.32 to 136.87 µg fly−1, respectively. Among Punjabi strains, D.G. Khan, Lodhran, Bahawalpur, Toba Tek Singh, Bahawalnagar, Rajanpur and Jhang strains exhibited very high levels of resistance (RR > 100) to methomyl; Bhakkar, Kasur, Vehari, Layyah, Muzaffargarh and R.Y. Khan showed high resistance (RR = 51–100 fold), while the Mianwali strain showed a moderate level of resistance to methomyl (RR = 36.45 fold). In case of Sindhi strains, very high levels of resistance (> 100 fold) were reported for Sukkar and Sanghar strains, high levels of resistance (RR 51–100 fold) for Khairpur, Jamshoro and Ghotki, and moderate resistance to methomyl (38.08 fold) in the Dadu strain. There was a significant synergism of methomyl toxicity in all field strains when methomyl bioassayed along with piperonyl butoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) providing clues of metabolic-based mechanisms of resistance to methomyl. In conclusion, insecticides used in crop farming can cause resistance development in non-target M. domestica. It is necessary to adopt the pest management activities that are safe for the environment and non-target insect species.

Insecticide resistance is a genetic change in response to continued selection pressure by insecticides that ultimately results in impaired chemical control in the field 12 . The development of resistance to insecticide in nontarget species could be more alarming in situations where the species is a major pest in another situation 13 . For instance, the presence of human-or animal-diseases vectors such as mosquitoes and flies would be non-target species in cropping areas, and if they developed resistance to insecticides, they become difficult to control when expended to nearby human populated areas [14][15][16] .
Pakistan is among the major cotton (Gossypium hirsutum L.) producing countries of the world, with around 2.79 million hectares cultivated area annually 17 . Cotton is mainly cultivated in the two major provinces of Pakistan i.e., Punjab and Sindh. Management of insect pests of cotton is important to ensure a high yield of the crop. Therefore, farmers heavily rely on the use of insecticides as a major insect pest management tool in both of the provinces 18,19 . Methomyl is among the most widely and commonly used insecticides for the management of a number of insect pests of cotton such as bollworms, armyworm, aphids, mealy bug, dusky bug, jassids and whiteflies 20,21 . It is a broad-spectrum insecticide and belongs to the carbamate class of insecticides. In Pakistan, it is being used usually in the form of spray applications for the last four decades, due to which resistance have been reported in different target insect species [21][22][23][24][25][26] . It is believed that the use of insecticides on crops also results in lethal and sublethal exposure to non-target insect species around farming areas as well 1,27 . Previously, resistance to mathomyl in the non-target mosquito Aedes albopictus (Skuse) was reported from the cotton fields of Punjab, Pakistan 28 . The house fly, Musca domestica Linnaeus, is a public health pest and one of the most common non-target insect species in cotton cultivated areas 29 . Musca domestica has been reported as one of pollinator and nectar feeder species of cotton crop [29][30][31][32][33] . In this way, it is expected that M. domestica get residues of methomyl after its application during flight, pollination and/or nectar feeding activities into the cotton fields, and develop resistance to methomyl after prolonged and repeated exposures.
Therefore, the present study was planned to check the hypothesis that the use of methomyl in cotton cultivation has caused resistance development in the non-target M. domestica collected from the cotton fields of major cotton producing areas of the Punjab and Sindh provinces of Pakistan.

Materials and methods
Musca domestica strains. Twenty field strains of M. domestica were collected from major cotton producing localities of the Punjab and Sindh provinces of Pakistan (Fig. 1) 34 . Cotton fields for M. domestica collection in the above localities were chosen based on history of methomyl use for the management of insect pests of cotton (personal communication with regional farmers and agriculture extension workers). An insecticide susceptible reference strain (Lab-susceptible) of M. domestica 35,36 was used in bioassays for the estimation of resistance to methomyl in field strains. Field strains were collected at the adult stage and brought to the laboratory of entomology, University of the Punjab, Lahore (31.5204° N, 74.3587° E) 34 . All strains were reared under the laboratory conditions (12:12 h dark/light photoperiod, 26 ± 2 °C, and 65 ± 5% relative humidity) following a well-established methodology using a sugarmilk-based diet 37,38 . Flies were reared in mesh cages, and pupae of specific date/time duration of a preceding generation were separated and kept in a new/empty cage for starting the subsequent generation. Adults from pupae usually emerged in 4-5 days; in this way flies of required age could easily be collected for bioassays. The first generation (F1) of field-collected strains was used for bioassays.
Bioassays and data analyses. Technical-grade methomyl (> 95% purity; Chem Service Inc, West Chester PA) was used for resistance detection bioassays in field strains of M. domestica. Topical bioassay method was used to apply methomyl doses on M. domestica as stated earlier in author's previous reports 15,39 : "Briefly, 0.5 μL of insecticide in acetone solution was applied by using a micropipette (0.1-2 µL, Acura ® manual 825, Socorex, Switzerland) on thoracic notum of 3-5-day-old female M. domestica. M. domestica were exposed to a range of methomyl doses that caused > 0 and < 100% mortality, and each bioassay was consisted of 20 M. domestica per dose. In the control treatment, flies were treated with acetone alone. Treated flies were kept in plastic jars (250 mL) provided with a cotton dental wick soaked with 20% sugar solution. All the bioassays were conducted at 26 ± 2 °C, 60 ± 5% RH, 12:12 (L/D) photoperiod, and replicated three times on different days. Mortality counts were made 48-h post-treatment and the data were analyzed by Probit analysis (Finney 1971) to determine median lethal doses (LD 50s ) of insecticides tested. Resistance ratios (RRs) were calculated by dividing LD 50 values of different field strains to those obtained with the Lab-susceptible reference strain, and categorized as high resistance (RR = 51-100 fold), moderate resistance (RR = 21-50 fold), low resistance (RR = 11-20 fold), very low resistance (RR = 2-10 fold) and no resistance (RR = 1)" 15,39 .
For synergism bioassays, M. domestica were exposed topically to enzyme inhibitors: piperonyl butoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) (Chem Service Inc, West Chester PA), with the maximum sublethal dose of 10 µg fly −1 , one hour before the insecticide treatment 40,41 . Treated M. domestica were then exposed to methomyl doses as stated above. Synergism ratio (SR) was calculated by dividing the LD 50 value of a particular strain to methomyl alone by the LD 50  www.nature.com/scientificreports/ or DEF. The SR value was considered significantly different if its 95% fiducial limit (FL) did not include "1" on the basis of the ratio test 42 .

Results
Field strains of M. domestica collected from different localities of the Punjab and Sindh provinces exhibited variable toxicity and resistance levels to methomyl compared with the Lab-susceptible strain ( Table 1) (Table 1).
Except the Lab-susceptible strain, toxicity of methomyl in all field strains increased significantly, based on non-overlapping 95% FL of LD 50 values and significant synergism ratios (SR), when bioassayed along with either PBO or DEF (Table 2). For instance, the LD 50 value of the Bhakkar strain reduced (toxicity increased) from 72.   (Table 2).

Discussion
It is a matter of great concern that the use of pesticides in agriculture often poses negative impact on non-target organisms, including insect species 43,44 . Based on the data of last few years, it has been observed that the use of insecticides in cropping systems resulted in the occurrence of resistance in the target and non-target insect species in Pakistan, which shows that this phenomenon is extremely widespread 1,15 . The present work could be considered as a continuation of our efforts to explore side-effects of insecticidal usage in agriculture on nontarget insect species, providing additional data of the impact of methomyl on M. domestica from localities not already explored. The data clearly indicates the occurrence of field-evolved resistance to methomyl in field strains of M. domestica in comparison to the Lab-susceptible strain. According to Valles et al. 45 , an insect strain should be assumed resistant to a particular insecticide if it shows more than tenfold RR value in comparison to the susceptible or reference strain. The data of the present study revealed that all of the field strains were resistant to methomyl and exhibited more than tenfold RR values in comparison to the Lab-susceptible strain. The susceptibility of reference strains of M. domestica to methomyl varies in different reports, depending upon strain origin, rearing conditions, and/or bioassay methods. While this does not undermine such studies, it is valuable to refer literature estimates as a rough means of comparison 46 . In the present study, the LD 50 value of methomyl for the Lab-susceptible strain (0.77 µg fly −1 ), is greater than UCR (0.58 µg fly −1 ) 46 , WHO (0.10 µg fly −1 ) 47 and Cooper (0.07 µg fly −1 ) 48 strains. Resistance ratio values for both Punjabi and Sindhi strains ranged from moderate to high levels, compared with the Lab-susceptible strain of M. domestica. Among Punjabi strains, D.G. Khan, Lodhran, Bahawalpur, Toba Tek Singh, Bahawalnagar, Rajanpur and Jhang strains exhibited very high levels of resistance (RR > 100) to methomyl; Bhakkar, Kasur, Vehari, Layyah, Muzaffargarh and R.Y. Khan showed high resistance (RR = 51-100 fold), while the Mianwali strain showed a moderate level of resistance to methomyl (RR = 36.45 fold). In case of Sindhi strains, very high levels of resistance (> 100 fold) were reported for Sukkar and Sanghar strains, high levels of resistance (RR 51-100 fold) for Khairpur, Jamshoro and Ghotki, and moderate resistance to methomyl (38.08 fold) in the Dadu strain. Resistance to methomyl could be due to the fact that field strains were collected from the cotton fields where methomyl was being used as one of the major insecticides to manage different insect pests such as bollworms, armyworm, aphids, mealy bug, dusky bug, jassids and whiteflies 20,49 . It is assumed that variation in resistance levels or toxicity in different strains might be due to www.nature.com/scientificreports/ the differences in origin of strains, climatic factors of collection sites and/or history of insecticide exposure. Variations in toxicity to insecticides due to these reasons have also been documented for different insect pests [50][51][52][53][54][55][56] . Variable levels of resistance to methomyl in M. domestica have been reported from different countries in the past 46,[57][58][59] . Previously, we also have reported low levels of resistance to methomyl in M. domestica strains collected from dairy farms in different localities, other than the ones in the present work, of Punjab, Pakistan 37 . Methomyl was used to target/manage M. domestica in dairy farms. However, in the present study, M. domestica strains were collected from the cotton fields where these are non-target species. Insecticidal usage in crops, besides controlling target pests, usually results in the lethal and sublethal exposures to non-target species that ultimately make these species resistant to insecticides with the passage of time 10,11 . Recently, resistance development has been reported in M. domestica and Aedes albopictus due to non-targeted exposure to insecticides used in rice farming 1,15 . Methomyl formulation has been registered in the form of emulsifiable concentrate (EC) and www.nature.com/scientificreports/ applied as sprays to manage different insect pests of cotton in Pakistan. Sprays of insecticides contaminate plant parts, soil, water and the surrounding air for a certain period of time 11,15 . It is believed that M. domestica get direct and/or indirect exposure to methomyl sprays during their routine life activities and developed resistance to methomyl as evidenced by the data of the present study.
Resistance to methomyl could be due to the activation of metabolic enzymes such as microsomal oxidases, esterases, etc., which can be initially checked by the use of enzyme inhibitors along with insecticides in bioassays 51,60 . Synergism of methomyl by PBO and DEF in Helicoverpa armigera (Hübner) 60 , M. domestica 61 and Oxycarenus hyalinipennis Costa 26 inferred that resistance may be attributable to microsomal oxidase and esterase detoxification. In the present work, synergism of methomyl with PBO and DEF in all the field strains was observed suggesting the possibility of metabolic mechanism of resistance. More in vitro studies are needed to further confirm the role of metabolic resistance mechanism in field strains of M. domestica.

Conclusion
The finding that non-target M. domestica has evolved resistance to methomyl used for the management of insect pests of cotton is troubling evidence of the side-effects of insecticidal usage in crop farming. The development of insecticide resistance in non-target species as a result of insecticide application against the targeted species usually lead to the outbreak of former species 11 . M. domestica is one of the major medical and veterinary pests and the development of resistance to insecticides may promote its outbreak coupled with an increased incidence of fly-borne diseases. Therefore, it is important to perform risk assessment studies in order to determine sideeffects of a particular insecticide on non-target species before and after its approval for use in cropping systems.

Data availability
The data presented in this study are available in article. www.nature.com/scientificreports/